We have earlier demonstrated for the first time that endocannabinoids acting via CB1 receptors promote voluntary alcohol drinking in an age-dependent manner, using a mouse model of two bottle/free choice paradigm (PNAS 100:1393, 2003). The brain-penetrant CB1 inverse agonist rimonabant used in this study was subsequently introduced as a treatment of obesity, but had to be wihdrawn from the pharmaceutical market in 2008, due to neuropsychiatric side effects, including anxiety, depression and suicidal ideation. An alternative approach, championed by my laboratory, was the introduction of peripherally restricted CB1 inverse agonists that retained the metabolic efficacy of rimonabant but were devoid of its neurobehavioral effects in rodent models of the metabolic syndrome. Paradoxically, such compounds were also found to reduce food intake, a centrally mediated effect. This paradox was resolved by the demonstration that peripheral CB1 blockade in diet-induced obese mice rapidly reversed their leptin resistance by reversing their hyperleptinemia, via inhibiting leptin production in adipose tissue and increasing leptin clearance in the kidney. This made us wonder whether the high alcohol preference of C57Bl6 mice, another central function promoted by CB1 activation, may also be affected indirectly through blockade of CB1 in the periphery. A possible mechanism involves ghrelin, a gastric peptide that promotes appetite via ghrelin receptors in the brain. Ghrelin also promotes alcohol craving and self-administration, as demonstrated recently in our Institute by Dr. Leggio and his group (Biol Psychiatry, 2014; Mol Psychiatry, 2017). The preliminary findings reported last year have been extended and the findings clearly indicate that rimonabant and the non brain-penetrant CB1 inverse agonists, JD5037, were equi-effective in markedly reducing total alcohol intake as well as ethanol preference in wild-type but not in CB1R-/- C57BL6 mice, using a 'two bottle, free choice' paradigm as well as a 'drinking in the dark' paradigm. Furthermore, peripheral CB1 blockade significantly reduced plasma levels of the biologically active acetylated ghrelin, with much less change in the level of its precursor, desacyl ghrelin, suggesting that the ghrelin acylation process, involving the enzyme GOAT and its substrates ghrelin and octanoate, maybe CB1R target(s). In additional experiments we found that both alcohol preference and absolute intake are lower in ghrelin knockout and ghrelin receptor1 (GHSR-1)knockout mice, with no additional reduction caused by peripheral CB1 receptor blockade. The effects on plasma ghrelin and the finding in the knockout strains are compatible with acylghrelin involvement in the effects of JD5037 on alcohol drinking. We also found that afferent vagal (sensory) denervation of the stomach also reduced alcohol drinking and drinking preference and abolished the efficacy of peripheral CB1 blockade as well as GHSR-1 blockade to reduce drinking. These findings are compatible with a scheme whereby alcohol drinking increases ghrelin acylation and/or acylghrelin secretion through CB1 receptor activation, and the released acylghrelin signals via ghrelin receptors (GHSR1) on vagal sensory terminals to promote ethanol drinking behavior. We next explored the cellular mechanism involved in the selective decrease in acyl-ghrelin production by peripheral CB1 blockade. Ghrelin-producing MGN3-1 stomach cells generate octanoic acid exclusively from long-chain fatty acid precursors, and also generate endocannabinoids and express CB1 receptors. JD5037 suppressed octanoyl-ghrelin but not desacyl-ghrelin production in MGN3-1 cells by reducing the levels of octanoyl-carnitine generated from palmityl-carnitine due to an increase in fatty acid oxidation (FAO), which was further confirmed by measuring FAO directly using a fluorescent FAO assay kit. Furthermore, treating MGN3-1 cells with selective inhibitors of 3 different enzymes in the FAO pathway selectively reduced acyl-ghrelin production and occluded the similar effect of JD5037. A manuscript summarizing these findings has been submitted for publication.